Diarrheal diseases remain a leading cause of deaths in children (~1.2 million/year) worldwide. In addition to its significant mortality and morbidity i developing countries, even in western countries, diarrhea associated with IBD and infection by food-borne pathogens or hospital acquired infections remains a major healthcare challenge. Therefore, better and novel treatment modalities are needed. In this regard, studies during our previous funding cycle provided pioneering in vitro and in vivo evidence for a potential probiotic Lactobacillus acidophilus (LA) based approach for the amelioration of diarrhea. We demonstrated that the mechanism of LA-mediated antidiarrheal effects involved stimulation of intestinal NaCl absorption, involving upregulation of the activities of NHE3 and DRA (the key intestinal apical membrane Na+/H+ and Cl-/HCO3- exchangers, respectively). Most importantly, the bacteria-free culture supernatant (CS) of LA was equally effective in stimulating NaCl absorption, defining a novel therapeutic modality to utilize bacteria-derived bioactive molecules, rather than live bacteria, for treating diarrheal disorders. Our studies further indicated that LA r LA- CS-induced enhancement of Cl-/HCO3- exchange activity involved increased levels of DRA on the apical membrane as well as its increased expression via transcriptional mechanisms. However, detailed cellular and molecular events governing LA- or LA-CS-induced alterations in DRA trafficking and regulation of DRA expression at the transcriptional level under normal physiology and under disease states are not known. Therefore, based on our published reports and strong preliminary data presented here we hypothesized that bacteria-free culture supernatant of L. acidophilus stimulates intestinal epithelial Cl-/HCO3- exchange activity via multiple mechanisms involving alterations of both intracellular trafficking of DRA as well as DRA expression. We also hypothesize that LA or LA-CS can combat the repression of DRA in models of IBD. We will test these hypotheses by systematic set of approaches utilizing both in vitro and in vivo models and state-of-the-art imaging and advanced molecular biology techniques. Studies proposed in Specific Aim 1 will investigate the mechanisms of LA-CS induced modulation of DRA trafficking in Caco2 cell monolayers and elucidate the role of lipid rafts, cytoskeletal and PDZ proteins; Specific Aim 2 will focus on elucidating mechanisms of LA-CS-induced stimulation of DRA transcription in Caco2 cells; and studies in Specific Aim 3 will examine the effects of short-term as well as long-term LA treatment on DRA function and membrane targeting mechanisms in in-vivo mouse models including IBD models. These studies will not only enhance our understanding of the mechanisms underlying proabsorptive/antidiarrheal role of probiotic LA but will also highlight the potential of the secreed bioactive molecules in LA-CS in combating the IBD associated diarrhea.